Modified p4hb

Abstract

The present disclosure relates to a nucleic acid construct that includes a modified prolyl 4-hydroxylase B subunit (P4HB) that has lost protein disulfide isomerase (PDI) activity, a composition that includes the nucleic acid construct, a plant body or isolated plant cells that include the nucleic acid construct, a use for the nucleic acid construct, a method for producing procollagen or collagen using a plant body or isolated plant cells that include the nucleic acid construct, procollagen or collagen produced by means of the production method, a composition that includes the procollagen or the collagen, and a method for preventing the death of a plant body or isolated plant cells.

Description

Modified P4HB

[0001] The present disclosure relates to a nucleic acid construct comprising a modified prolyl 4-hydroxylase B subunit (P4HB) deficient in protein disulfide isomerase (PDI) activity, a composition comprising the nucleic acid construct, a plant or isolated plant cell comprising the nucleic acid construct, use of the nucleic acid construct in a method for producing procollagen or collagen, a method for producing procollagen or collagen using a plant or isolated plant cell comprising the nucleic acid construct, procollagen or collagen produced by the production method, and a composition comprising the procollagen or collagen.

[0002] Advantages of plant-expressed collagen: Collagen is one of the proteins that mainly make up the dermis, ligaments, tendons, bones, and cartilage of vertebrates, and is a major component of the extracellular matrix. It is also used as a pharmaceutical ingredient and a medium component for cell culture. The main source of collagen for industrial use is livestock such as cows and pigs, but using it as a pharmaceutical ingredient carries the risk of zoonotic infections and allergies. Producing human collagen in a plant expression system can avoid these risks.

[0003] Plants expressing collagen chains are known in the art and have been reported in, for example, US Pat. No. 6,617,431 (Patent Document 1), Merle et al., 2002 (Non-Patent Document 1), and Ruggiero et al., 2000 (Non-Patent Document 2). However, prolyl hydroxylases in plant cells lack the appropriate substrate sequence specificity, and therefore post-translational modification of collagen in plants requires co-expression of prolyl 4-hydroxylase (P4H: E.C. 1.14.11.2) (Olsen et al., 2003: Non-Patent Document 3).

[0004] P4H is a dimer or tetramer of two subunits, A and B (P4HA and P4HB). The prolyl hydroxylase active center of P4H is present in P4HA, but P4HA alone is insoluble and tends to aggregate, and it must form a complex with P4HB to exert its activity. In contrast, P4HB is a multifunctional protein that not only contributes to the solubility of P4H, but also functions alone as a protein disulfide isomerase (PDI) and is involved in the formation of SS bonds in proteins. Human P4HB consists of 508 amino acid residues, with two PDI active centers consisting of the Cys-Gly-His-Cys sequence located at positions 53-56 and 397-400. It is known that the oxidation-reduction of two cysteine ​​residues in the active center is involved in PDI activity. Previous studies have reported that PDI activity is reduced or lost by substituting one cysteine ​​residue in one or both of the two active centers of P4HB with a serine residue, and that a complex of the mutant P4HB with P4HA exhibits P4H activity in vitro using 2-oxo[1-C]glutarate as a substrate (Vuori et al., 1992: Non-Patent Document 4). Other previous studies have reported that expression of P4H as a fusion protein with a soluble partner such as GST catalyzes the hydroxylation of a peptide model substrate in vitro (JP Patent Publication No. 2022512534: Patent Document 2). However, it has not been confirmed that P4HB lacking PDI activity or a P4H complex containing this P4HB performs the appropriate post-translational modification of collagen, i.e., hydroxylation of specific proline residues, in plant cells.

[0005] The inventors of the present disclosure have previously found that transient expression of collagen in plants suppresses N-terminal cleavage of procollagen (Japanese Patent Application No. 2022-123600). However, when attempts were made to co-express type I collagen and P4H in a plant transient expression system, it was found that severe necrosis occurred in the plant. Therefore, an improved method for producing collagen in plants was needed.

[0006] US6617431 special table 2022-512534

[0007] Merle et al. , FEBS Letter, 2002, Vol. 515, Issue 1-3, 2002, pp. 114-118 Ruggiero et al. , FEBS Letter, 2000, Vol. 469, Issue 1, pp. 132-6 Olsen et al. , Adv Drug Deliv Rev, 2003 Nov 28;55(12), pp. 1547-67Vuori et al. , The EMBO Journal, 1992, vol. 11 no. 11, pp. 4213-4217

[0008] An object of the present disclosure is to provide a new method for producing procollagen or collagen using plants.

[0009] The present inventors attempted to co-express type I collagen and P4H in a plant transient expression system, and found that severe necrosis occurred in the plant. After diligently investigating the cause, they discovered that the B subunit of P4H (P4HB) was the cause of plant necrosis. After searching for a way to solve this problem, the present inventors created a P4HB with a PDI activity-loss mutation by modifying the PDI active center of P4HB, and found that co-expression with P4HA or expression of a fusion protein with P4HA in a plant transient expression system could suppress plant necrosis. Furthermore, they confirmed that co-expression with collagen allowed the P4H complex containing the mutant P4HB to function as a collagen post-translational modification enzyme in plants, and found that collagen with post-translational modifications and excellent thermostability could be produced in a plant transient expression system.

[0010] Based on the above findings, the present disclosure provides a nucleic acid construct comprising a modified prolyl 4-hydroxylase B subunit (P4HB) that lacks PDI activity, a composition comprising the nucleic acid construct, a plant or isolated plant cell comprising the nucleic acid construct, use of the nucleic acid construct in a method for producing procollagen or collagen, a method for producing procollagen or collagen using a plant or isolated plant cell comprising the nucleic acid construct, procollagen or collagen produced by the production method, and a composition comprising the procollagen or collagen.

[0011] In one aspect, the disclosure provides a nucleic acid construct having expression control elements functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB lacks protein disulfide isomerase (PDI) activity.

[0012] In one aspect, the disclosure provides a composition comprising a nucleic acid construct having expression control elements functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB lacks protein disulfide isomerase (PDI) activity.

[0013] In one aspect, the disclosure provides a plant or isolated plant cell comprising a nucleic acid construct having expression control elements functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity.

[0014] In one aspect, the disclosure provides use of a nucleic acid construct, or a composition comprising the nucleic acid construct, in a method for producing procollagen or collagen from a plant or isolated plant cell, comprising an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB lacks protein disulfide isomerase (PDI) activity.

[0015] In one aspect, the present disclosure provides a method for producing procollagen or collagen, comprising the steps of: A) providing a plant or isolated plant cell containing a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell.

[0016] In one aspect, the present disclosure provides procollagen or collagen produced by a method for producing procollagen or collagen, the method comprising the steps of: A) providing a plant or isolated plant cell containing a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell.

[0017] More specifically, the present disclosure provides the following: [Item 1] A nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB lacks protein disulfide isomerase (PDI) activity. [Item 2] The nucleic acid construct of Item 1, wherein the P4HB encoded by the nucleic acid sequence has the function of post-translationally modifying procollagen or collagen in a plant cell that expresses P4HA. [Item 3] The nucleic acid construct of Item 1, wherein the P4HB encoded by the nucleic acid sequence has an amino acid substitution in the PDI active center of the subunit. [Item 4] The nucleic acid construct of Item 1, wherein the P4HB encoded by the nucleic acid sequence is fused to a prolyl 4-hydroxylase A subunit. [Item 5] The nucleic acid construct of Item 1, wherein the plant is a Nicotiana plant. [Item 6] A composition comprising the nucleic acid construct of Item 1. [Item 7] A plant or isolated plant cell comprising the nucleic acid construct of Item 1. [Item 8] Use of the nucleic acid construct of any one of Items 1 to 5, the composition of Item 6, or the plant or isolated plant cell of Item 7 in a method for producing procollagen or collagen from a plant or isolated plant cell. [Item 9] A method for producing procollagen or collagen, comprising: A) providing a plant or isolated plant cell containing the nucleic acid construct of any one of Items 1 to 5, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell. [Item 10] The method of Item 9, further comprising: C) isolating the procollagen or collagen secreted extracellularly by step B. [Item 11] Procollagen or collagen produced by the method of Item 9.[Item 12] A method for preventing the withering of a plant or an isolated plant cell, comprising: A) introducing a nucleic acid encoding collagen or procollagen into a plant or an isolated plant cell; and B) introducing the nucleic acid construct of any one of Items 1 to 5 and a nucleic acid encoding P4HA into the plant or isolated plant cell.

[0018] The present disclosure has the effect of providing a nucleic acid construct comprising a modified P4HB deficient in protein disulfide isomerase activity, use of the nucleic acid construct in a method for producing procollagen or collagen from a plant or isolated plant cell, a method for producing procollagen or collagen from a plant or isolated plant cell using the nucleic acid construct, a composition containing the nucleic acid construct, and a modified P4HB protein encoded by the nucleic acid construct.

[0019] FIG. 1 is a schematic diagram showing the structure of a collagen gene expression cassette. FIG. 2 is a schematic diagram showing the structure of a P4HA and P4HB modifying enzyme expression vector. FIG. 3 is a schematic diagram showing the structure of an expression cassette for a fusion protein of P4HA and modified P4HB, or P4HA and a water-soluble protein (MBP or GFP). FIG. 4 is a schematic diagram showing the structure of an expression cassette for RNA silencing suppressor p19 derived from Tomato bushy stunt virus. FIG. 5 is a schematic diagram showing the amino acid sequence of the PDI active center modified in modified P4HB. FIG. 6 is a photograph showing the state of a plant in which modified P4HB has been expressed. A shows the state of a plant inoculated with P4HB in which only one PDI active center has been modified, and B shows the state of a plant inoculated with P4HB in which both PPI active centers have been modified. FIG. 7 shows the results of a test to confirm collagen expression in plants expressing collagen α1 alone or modified P4HB. The arrow indicates the collagen α1 band. FIG. 8 shows the results of a test to confirm the state of collagen after trypsin treatment following heat treatment at 33°C for extracts from plants expressing collagen α1 alone or modified P4HB. FIG. 9 shows the results of a test to confirm the state of collagen after trypsin treatment following heat treatment under gradient conditions for extracts from plants expressing collagen α1 alone or modified P4HB (human P4HA / human P4HB_NC-AA fusion protein). FIG. 10 shows the results of a test to confirm the state of collagen after trypsin treatment following heat treatment under gradient conditions for extracts from plants expressing modified P4HB (human P4HA + human P4HB_NC-SS co-expression) or (nematode P4HA / mouse P4HB_NC-SS fusion). 11 shows the results of testing collagen trypsin resistance in plants expressing P4HA fused with another water-soluble protein instead of P4HB. The arrow indicates the collagen α1 band.

[0020] The inventors of the present disclosure speculated that necrosis was caused by interference of the PDI activity of P4HB with disulfide bond formation in plant proteins, and created a PDI activity-loss mutant P4HB by modifying the PDI active center of P4HB. Co-expression of this mutant plant with P4HA or expression of a fusion protein with P4HA in a transient expression system successfully suppressed necrosis. Furthermore, they found that co-expression with type I collagen enabled it to function as a collagen post-translational modification enzyme in plants.

[0021] DEFINITIONS Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0022] In this specification, when multiple ranges of numerical values ​​are shown, the same applies to ranges formed by combining any lower limit value and upper limit value of those multiple ranges.

[0023] As used herein, the term "substantially" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, but is intended to encompass a desired state and a state that is unavoidably not achieved due to biological or chemical properties, taking into consideration, for example, that a biological or chemical phenomenon may not completely achieve a desired state.

[0024] As used herein, the term "about" in connection with a numerical value means that the value can vary within, for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0025] As used herein, the term "comprise" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure belongs, and includes, for example, "comprises" and "consists of." Specifically, a composition "comprising" A may contain another component, B, in addition to containing only A.

[0026] As used herein, the terms "consisting of" or "composed of" when referring to a composition have the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains, but are used to indicate components that exclusively constitute the composition. For example, a composition "consisting of" A contains exclusively A. However, in one embodiment, a composition "consisting of" A encompasses an embodiment in which a contaminant other than A is contained that is unavoidable in production due to biological and chemical properties.

[0027] As used herein, the term "deficiency" has the same meaning as commonly understood by those skilled in the art to which this disclosure pertains, but typically means that part or all of an inherent function or physical configuration has been lost.

[0028] In the present disclosure, the "identity" of an amino acid sequence or a nucleotide sequence has the meaning commonly understood by those skilled in the art to which the present disclosure pertains. Typically, the two amino acid sequences to be compared are aligned so that as many amino acid or nucleotide sequences as possible are identical, and the number of identical amino acids or nucleotides is divided by the total number of amino acids or nucleotides, expressed as a percentage. During the alignment, gaps may be inserted as needed into one or both of the two sequences being compared. Such sequence alignment can be performed using well-known programs such as BLAST, FASTA, and CLUSTAL W. When gaps are inserted, the total number of amino acids or nucleotides is calculated by counting one gap as one amino acid or nucleotide. If the total number of amino acids or nucleotides counted in this way differs between the two sequences being compared, the identity (%) is calculated by dividing the number of identical amino acids or nucleotides by the total number of amino acids or nucleotides in the longer sequence. However, when the sequence to be compared is linked to any other sequence, only the corresponding region is extracted and compared to calculate the identity.

[0029] In the present disclosure, "transient" protein expression has the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains, but typically means that the protein is expressed in a host cell without the nucleic acid sequence encoding the protein being integrated into the genome of the host cell. Such transient expression can be achieved, for example, by using a viral vector.

[0030] In the present disclosure, the term "fusion protein" has the meaning commonly understood by those skilled in the art to which the present disclosure pertains, but typically refers to a single protein obtained by fusing at least a portion of a first protein to at least a portion of a second protein. In one embodiment, a heterologous amino acid sequence may be included as a linker between at least a portion of the first protein and at least a portion of the second protein. Methods for producing such fusion proteins are known. For example, a fusion protein can be obtained by linking a portion of a nucleic acid encoding at least a portion of the first protein to a nucleic acid encoding at least a portion of the second protein in translation frame to create a fusion gene, and then expressing the fusion gene in an appropriate protein expression system.

[0031] In the present disclosure, the term "expression control element" has the meaning commonly understood by those skilled in the art to which this disclosure pertains, but typically refers to a non-gene coding sequence located upstream or downstream of a gene that controls the expression of the gene. Such an expression control element may sometimes be referred to as a 5' untranslated region (5'UTR) or a 3' untranslated region (3'UTR). In one embodiment, the expression control element includes a transcriptional control element and a translational control element. Examples of transcriptional control elements include, but are not limited to, transcriptional promoters, transcriptional enhancers, and transcriptional terminators. Examples of translational control elements include, but are not limited to, translational enhancers.

[0032] In the present disclosure, the expression control element and the gene whose expression is to be controlled are "operably linked" in the sense commonly understood by those skilled in the art. Typically, the expression control element and the gene nucleic acid sequence controlled by the expression control element are arranged in such a way that the expression control element can perform its intended function and control the expression of the gene nucleic acid sequence. Expression control can be achieved by relying on the arrangement relative to the sense or antisense strand of the gene nucleic acid sequence. For this purpose, direct chemical linkage is not necessarily required. In one embodiment, an expression control element, such as a transcription enhancer sequence, can be located at a distance from the gene nucleic acid sequence in the same nucleic acid molecule as the gene nucleic acid sequence to be controlled. In another embodiment, the expression control element can be located on a nucleic acid molecule different from the nucleic acid molecule in which the gene nucleic acid sequence to be controlled is located. In one embodiment, when the expression control element is a transcription promoter sequence, the gene nucleic acid sequence to be controlled is located downstream (3' direction) of the transcription promoter sequence, and the two sequences are optionally covalently linked to each other via one or more base sequences. The distance between the promoter sequence and the nucleic acid sequence to be recombinantly expressed is preferably less than 200 base pairs, particularly preferably less than 100 base pairs, and very particularly preferably less than 50 base pairs. Conventional recombination and cloning techniques can be used (e.g., Maniatis T, Fritsch EF and Sambrook J (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor (NY)).

[0033] In the present disclosure, the term "nucleic acid construct" has the meaning commonly understood by those skilled in the art to which the present disclosure pertains, and typically refers to a nucleic acid molecule comprising one or more nucleic acid sequences having a certain function. For example, in one embodiment, the nucleic acid construct comprises one or more protein-encoding genes, expression control elements, etc. The nucleic acid construct may be a DNA molecule or an RNA molecule.

[0034] As used herein, "isolated" has the meaning commonly understood by those skilled in the art to which this disclosure pertains, typically meaning that it is removed by hand from its natural environment by a process involving human manipulation and is not a product of nature. In one embodiment, an isolated substance or molecule, such as a nucleic acid molecule or a protein molecule, can exist in a purified form. In one embodiment, an isolated substance or molecule can exist in a non-natural environment, such as a transgenic host cell. In one embodiment, an isolated substance or molecule exists separated from some or all of the coexisting materials in the natural state. For example, a polynucleotide present as part of a vector, or a naturally occurring substance coexisting with non-naturally occurring materials in a composition, is in an isolated state.

[0035] (Nucleic Acid Construct of the Present Disclosure) In one aspect, the present disclosure provides a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B is deficient in protein disulfide isomerase (PDI) activity.

[0036] An expression control element functional in plant cells used in the nucleic acid construct of the present disclosure refers to an expression control element that substantially accomplishes its intended function in plant cells, and is an expression control element that controls the expression of a gene of interest in a plant organism as intended. Examples of expression control elements include, but are not limited to, barley amylase signal peptide (Barley AMY 1.2 Signal Peptide, DNA sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2), CaMV 35S promoter (SEQ ID NO: 5), Arabidopsis thaliana ADH-derived 5'-UTR (SEQ ID NO: 6), TMV omega sequence (SEQ ID NO: 7), and HSP terminator (SEQ ID NO: 8).

[0037] Examples of expression control elements functional in plant cells for use in the nucleic acid constructs of the present disclosure include Nicotiana, bryophytes (e.g., P. patens), potato (e.g., S. tuberosum), grasses (e.g., O. sativa), Brassicaceae, and lettuce, with Nicotiana being preferred. Examples of Nicotiana plants that are functional in plant cells include N. benthamiana, N. tabacum, and N. excelsior, but these are not limiting examples.

[0038] PH4B encoded by the sequence contained in the nucleic acid construct of the present disclosure is a protein well known in the technical field of the present disclosure, and its amino acid sequence and the gene nucleic acid sequence encoding the protein in each organism are publicly known. P4HB used in the present disclosure can be derived from various organisms, and the type is not particularly limited as long as the objectives of the present disclosure can be achieved. For example, P4HB from primates such as humans and monkeys, mammals such as mice, rats, dogs, cats, cows, pigs, and camels, birds, reptiles, amphibians, fish, and invertebrates such as nematodes can be used, but is not limited to these.

[0039] The P4HB encoded by the sequence contained in the nucleic acid construct of the present disclosure lacks PDI activity. In one embodiment, the PDI activity of the P4HB contained in the nucleic acid construct of the present disclosure, when collagen is used as a substrate, is 0.5 to 70%, 0.5 to 60%, 0.5 to 50%, 0.5 to 40%, 0.5 to 30%, 0.5 to 20%, 0.5 to 10%, 0.5 to 5%, 0.5 to 1%, or 0.5% or less compared to the PDI activity of native P4HB. Methods for measuring the PDI activity of P4HB are known to those skilled in the art; for example, the method described in Vuori et al., The EMBO Journal, 1992, vol. 11 no. 11, pp. 4213-4217 (Non-Patent Document 4) can be used with modifications.

[0040] In one embodiment, P4HB lacking PDI activity encoded by a sequence contained in a nucleic acid construct of the present disclosure has at least one amino acid mutation in at least one PDI active center. The amino acid sequence regions constituting the PDI active center of a P4HB protein are known to those skilled in the art. For example, in the case of human P4HB, the two PDI active centers are respectively constituted by the amino acid sequence regions at positions 53-56 and 397-400. For example, each PDI active center of human P4HB has two cysteine ​​residues. The amino acid mutation is not particularly limited and includes amino acid substitution, deletion, and insertion.

[0041] In one embodiment, P4HB lacking PDI activity encoded by a sequence contained in a nucleic acid construct of the present disclosure has at least one cysteine ​​residue substituted with another amino acid residue in at least one PDI active center. In one embodiment, preferably, P4HB lacking protein disulfide isomerase (PDI) activity contained in a nucleic acid construct of the present disclosure has a cysteine ​​residue at the N-terminus of each PDI center substituted with another amino acid residue. The amino acid residue substituted for the cysteine ​​residue is not particularly limited as long as it does not form a disulfide bond with another amino acid. Natural or unnatural amino acid residues can be used as the amino acid residue substituted for the cysteine ​​residue. In one embodiment, preferably, P4HB lacking protein disulfide isomerase (PDI) activity contained in a nucleic acid construct of the present disclosure has a cysteine ​​residue at the N-terminus of each PDI center substituted with an alanine residue, a serine residue, or a histidine residue.

[0042] In one aspect, P4HB lacking PDI activity, encoded by a sequence contained in a nucleic acid construct of the present disclosure, has the function of post-translationally modifying procollagen or collagen, i.e., hydroxylating specific proline residues, in plant cells expressing P4HA (Olsen et al., 2003: Non-Patent Document 3). When expressed in plant cells together with P4HA, P4HB lacking protein disulfide isomerase activity of the present disclosure forms a complex, which can post-translationally modify procollagen or collagen in the plant cells. In one aspect, P4HB lacking PDI activity encoded by a sequence contained in a nucleic acid construct of the present disclosure is co-expressed with P4HA in cells. This post-translational modification can suppress procollagen or collagen from being degraded by proteolytic enzymes. In one aspect, P4HB lacking protein disulfide isomerase activity of the present disclosure post-translationally modifies procollagen or collagen in plant cells, thereby improving the thermal stability of the procollagen or collagen. In one aspect, procollagen or collagen post-translationally modified in plant cells by P4HB deficient in protein disulfide isomerase activity of the present disclosure exhibits a higher affinity for procollagen or collagen at high temperatures, for example, at about 28°C or higher, about 29°C or higher, about 30°C or higher, about 31°C or higher, about 32°C or higher, about 33°C or higher, about 34°C or higher, about 35°C or higher, or about 36°C or higher, compared to procollagen or collagen not having the modification. , about 37°C or higher, about 38°C or higher, about 39°C or higher, about 40°C or higher, about 41°C or higher, about 42°C or higher, or 28°C to 42°C, 29°C to 42°C, 30°C to 42°C, 31°C to 42°C, 32°C to 42°C, 33°C to 42°C, 34°C to 42°C, 35°C to 42°C, 36°C to 42°C, 37°C to 42°C, 38°C to 42°C, 39°C to 42°C, 40°C to 42°C, and 41°C to 42°C. The improvement in the thermal stability of procollagen or collagen due to post-translational modification can be confirmed by known analytical methods. For example, this can be confirmed by thermally denaturing a plant cell extract containing the procollagen or collagen at room temperature or higher, followed by analyzing the degradation products obtained by trypsin digestion.

[0043] In one embodiment, the P4HB lacking PDI activity encoded by the sequence contained in the nucleic acid construct of the present disclosure has an amino acid mutation unrelated to PDI activity. In one embodiment, the P4HB lacking PDI activity encoded by the sequence contained in the nucleic acid construct of the present disclosure has about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95%, about 96%, about 97%, or about 99% or more amino acid sequence identity with native P4HB. The P4HB lacking PDI activity encoded by the sequence contained in the nucleic acid construct of the present disclosure may contain one or more amino acid substitutions, insertions, or deletions relative to native P4HB.

[0044] In one aspect, P4HB lacking PDI activity, encoded by a sequence contained in a nucleic acid construct of the present disclosure, may be expressed as a fusion protein fused with a heterologous protein. Methods for producing heterologous proteins are known to those skilled in the art. For example, a fusion protein of P4HB and a heterologous protein can be expressed by including a nucleic acid sequence encoding a fusion protein of P4HB and a heterologous protein in the nucleic acid construct of the present disclosure. The manner in which P4HB and a heterologous protein are fused is not particularly limited, and the positional relationship between the nucleic acid sequence encoding part or all of P4HB and the nucleic acid sequence encoding part or all of the heterologous protein, the presence or absence of a linker sequence, and the like can be appropriately selected by those skilled in the art depending on the purpose.

[0045] In one embodiment, P4HB lacking PDI activity encoded by a sequence contained in a nucleic acid construct of the present disclosure is expressed as a fusion protein in which P4HB is fused with P4HA, preferably in which the full-length P4HB protein is fused to the C-terminus of the full-length P4HA protein via a linker (GSGSGRITMLSRALLCLALAWAARVGA).

[0046] The nucleic acid constructs of the present disclosure can be prepared by conventional chemical synthesis methods known to those skilled in the art or by genetic engineering techniques. For example, they can be synthesized by synthesizing a cDNA having a naturally occurring sequence, and then using this cDNA as a template to introduce appropriate mutations using appropriate primers. Alternatively, the nucleic acid construct to be synthesized can be divided into several short regions, and multiple DNA fragments can be chemically synthesized. The fragments can then be linked using the known fusion PCR method to synthesize a DNA construct. RNA constructs can be prepared by transcription from the DNA construct.

[0047] Any method known to those skilled in the art to which the present disclosure pertains can be used to introduce a mutation into the sequence of a nucleic acid construct. For example, those skilled in the art can use site-directed mutagenesis methods such as the Kunkel method and the gapped duplex method, overlap extension PCR, and QuikChange, but are not limited to these.

[0048] The nucleic acid construct of the present disclosure may be DNA, RNA, or any analog thereof known to those skilled in the art to which the present disclosure pertains. For example, the nucleic acid construct of the present disclosure may contain unnatural bases, and may also contain a hydrocarbon chain or a peptide chain as part thereof.

[0049] In one aspect, the nucleic acid construct of the present disclosure is provided as an isolated nucleic acid molecule. In one aspect, the nucleic acid construct of the present disclosure is provided as a nucleic acid molecule consisting solely of the nucleic acid construct. In one aspect, the nucleic acid construct of the present disclosure is provided in a state where it is further bound to other nucleic acids. For example, it is provided as an expression vector or an amplification vector inserted into a vector. In one aspect, the nucleic acid construct of the present disclosure is provided as a recombinant virus. The recombinant virus can be produced by conventionally modifying any plant virus vector known to those skilled in the art to which the disclosure pertains (e.g., tobacco mosaic virus, potato virus X, geminivirus, etc.; see "Proteins, Nucleic Acids, Enzymes," vol. 45, pp. 607-613, etc.). In one embodiment, the nucleic acid construct of the present disclosure is provided as a transformation vector for Agrobacterium-mediated plant transformation by the binary vector method (R. Nishiguchi, et al., Molecular and General Genetics, 1987, Vol. 206, pp. 1-8). In one embodiment, it is preferably provided in a state where it is inserted into the transformation vector pRI 201-AN (Takara Bio).

[0050] In one embodiment, the nucleic acid construct of the present disclosure is provided as a composition in which it is further mixed with other nucleic acid molecules and / or substances other than nucleic acids, such as a nucleic acid molecule having a nucleic acid sequence encoding PH4A, a buffer solution for improving the storage stability of the nucleic acid construct, etc.

[0051] In one aspect, the nucleic acid construct of the present disclosure is provided as a cell containing the nucleic acid construct. The type of cell is not particularly limited and can be appropriately selected depending on the purpose, such as genetically modifying, amplifying, or expressing the nucleic acid construct within the cell. For example, the nucleic acid construct can be provided as a plant cultured cell transformed with the nucleic acid construct for the purpose of expressing the nucleic acid construct, or as a bacterial cell transformed with the nucleic acid construct for genetically modifying or amplifying the nucleic acid construct. In these cells, the nucleic acid construct of the present disclosure may or may not be integrated into the genomic DNA of the cell. In one aspect, the nucleic acid construct of the present disclosure is provided as a plant containing the nucleic acid construct. The plant may be one transfected with the nucleic acid construct of the present disclosure or a progeny of such a transfected plant.

[0052] (Composition Comprising a Nucleic Acid Construct of the Present Disclosure) In one aspect, the present disclosure provides a composition comprising a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B lacks protein disulfide isomerase (PDI) activity. In one aspect, the composition of the present disclosure comprises, in addition to the nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B lacks protein disulfide isomerase (PDI) activity, another nucleic acid molecule and / or a non-nucleic acid molecule. For example, the nucleic acid construct of the present disclosure can be provided as a composition in combination with a nucleic acid molecule comprising a nucleic acid sequence encoding PH4A, a nucleic acid molecule encoding another protein that contributes to stable expression of PH4A in plant cells (such as Tomato bushy stunt virus-derived RNA silencing suppressor p19 (UniProtKB-P50628)), a buffer solution for improving the storage stability of the nucleic acid construct, an antioxidant, and other components.

[0053] In one aspect, a composition comprising a nucleic acid construct of the present disclosure is provided as a composition for use in the manufacture of a plant or plant cell that produces collagen or procollagen.

[0054] (Plant or isolated plant cell containing a nucleic acid construct of the present disclosure) In one aspect, the present disclosure provides a plant or isolated plant cell containing a nucleic acid construct of the present disclosure. The plant or isolated plant cell may be obtained by introducing a nucleic acid construct into the plant or plant cell, or by growing or multiplying the plant or plant cell. In one aspect, the plant or isolated plant cell containing the nucleic acid construct of the present disclosure may be a subculture of a plant that has been introduced with the nucleic acid construct.

[0055] (Use of the nucleic acid construct of the present disclosure or a composition comprising the nucleic acid construct) In one aspect, the present disclosure provides the use of a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B lacks protein disulfide isomerase (PDI) activity, or a composition comprising the nucleic acid construct, in a method for producing procollagen or collagen from a plant or isolated plant cell.

[0056] In one embodiment, the nucleic acid construct described above (Nucleic acid construct of the present disclosure) can be used as the nucleic acid construct utilized in the use of the present disclosure. In one embodiment, the composition described above (Composition comprising the nucleic acid construct of the present disclosure) can be used as the composition utilized in the use of the present disclosure.

[0057] In one aspect, a use of the present disclosure comprises (A) transforming a plant or isolated plant cell with a nucleic acid construct of the present disclosure or a composition comprising a nucleic acid construct, and / or (B) expressing P4HB and P4HA, as well as transiently expressing procollagen, in a plant or isolated plant cell transformed with a nucleic acid construct of the present disclosure or a composition comprising a nucleic acid construct.

[0058] In one embodiment, the use of the present disclosure further comprises the step of isolating the extracellularly secreted procollagen or collagen.

[0059] The use of the present disclosure provides for the production of procollagen or collagen with excellent heat stability using a plant or isolated plant cells, and provides collagen that is not contaminated by viruses or other infectious agents of animal origin and can be used in the pharmaceutical, therapeutic, cosmetic, food, and other fields.

[0060] (Method for Producing Procollagen or Collagen Using a Plant or Isolated Plant Cell Comprising a Nucleic Acid Construct of the Present Disclosure) In one aspect, the present disclosure provides a method for producing procollagen or collagen using a plant or isolated plant cell comprising a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity.

[0061] In one embodiment, the method for producing procollagen or collagen of the present disclosure comprises the following steps: A) providing a plant or isolated plant cell containing a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell.

[0062] In the above-mentioned step B) "step of transiently expressing procollagen in a plant or isolated plant cells," an expression vector such as a viral vector containing a nucleic acid sequence encoding procollagen is typically expressed in a plant or isolated plant cells without being integrated into the genome of the host cell.

[0063] Transient expression of procollagen makes it possible to suppress N-terminal cleavage in plant cells. Transient expression of procollagen in a plant or isolated plant cells can be carried out by methods known to those skilled in the art in the technical field to which the present disclosure pertains, and the expression level, expression period, etc. of procollagen can be appropriately adjusted by those skilled in the art by appropriately adjusting the vector, promoter, medium composition, culture conditions, etc. used, within the scope of the present disclosure.

[0064] For example, when a transformation vector is used for transient expression, a large amount of collagen is produced in a plant or isolated plant cells after vector inoculation. For example, when collagen is produced using a plant, the plant can be cultivated for about 3 to 15 days after vector inoculation, and then collagen or procollagen can be extracted and recovered from plant tissues such as leaves. For example, the plant can be cultivated for about 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, or 10 to 15 days after vector inoculation, and then collagen or procollagen can be extracted and recovered from plant tissues such as leaves.

[0065] For example, when a recombinant virus is used, the virus grows in a plant infected with the virus or in isolated plant cells, resulting in the production of a large amount of collagen in the plant cells. For example, when collagen is produced using a plant, the plant is cultivated for about 3 to 15 days after inoculation with the virus, and collagen or procollagen can then be extracted and recovered from plant tissues such as leaves.

[0066] In one aspect, the method for producing procollagen or collagen of the present disclosure includes a step C) of separating the procollagen or collagen secreted extracellularly by step B. Any method known to those skilled in the art to which the present disclosure pertains can be used in this step. For example, the methods described in C. D. Mount et al., Archives of Biochemistry and Biophysics, 240:33 (1985) and U. H. Gregory and I. R. Willshire, Hoppe-Sayler's Z. Physiol. Chem., 356:1765 (1975) can be used, but are not limited to these. More specifically, for example, when collagen or procollagen is produced by expressing collagen in a tobacco plant, frozen and stored tobacco plant leaves can be ground in an appropriate buffer, and collagen or procollagen can be extracted and purified from the buffer by salting out.

[0067] In one aspect, the plants or isolated plant cells described above (Plants or isolated plant cells containing a nucleic acid encoding 4HB and a nucleic acid encoding collagen or procollagen) used in the method for producing procollagen or collagen of the present disclosure can be the plants or isolated plant cells described above (Plants or isolated plant cells containing a nucleic acid construct of the present disclosure), and can be produced using the nucleic acid construct described above (Nucleic acid construct of the present disclosure).

[0068] The step of expressing PH4B in a plant or isolated plant cells can be carried out using methods known in the technical field of the present disclosure. In one embodiment, PH4B is transiently expressed in the method for producing procollagen or collagen of the present disclosure. Methods known in the technical field of the present disclosure can be used to transiently express PH4B in a plant or isolated plant cells. For example, transient expression of procollagen from a plant or isolated plant cell can be achieved by infecting the plant or isolated plant cell with a nucleic acid encoding PH4B as an expression vector. If the virus is an RNA virus, the nucleic acid construct of the present disclosure is RNA; if the virus is a DNA virus, the nucleic acid construct of the present disclosure is DNA. In one embodiment, PH4B can be transiently expressed by transforming Agrobacterium tumefaciens with a vector plasmid and then infecting the Agrobacterium into a plant or isolated plant cell.

[0069] The P4HA used in the method for producing procollagen or collagen of the present disclosure is a well-known protein in the technical field of the present disclosure, and its amino acid sequence and the gene nucleic acid sequence that encodes this protein in each organism are known.The P4HA used in the present disclosure can be derived from various organisms, and its type is not particularly limited as long as it can achieve the purpose of the present disclosure.For example, the P4HA of mammals such as humans, monkeys, etc., primates, mice, rats, dogs, cats, cows, pigs, camels, etc., birds, reptiles, amphibians, fish, nematodes, etc., invertebrates can be used, but is not limited thereto.

[0070] The P4HA and P4HB used in the method for producing procollagen or collagen of the present disclosure may be derived from the same organism or from different organisms, such as, but not limited to, a combination of human P4HA and human P4HB, or a combination of nematode P4HA and mouse P4HB.

[0071] In one embodiment, the P4HA used in the method for producing procollagen or collagen of the present disclosure may have amino acid mutations.In one embodiment, the P4HA used in the method for producing procollagen or collagen of the present disclosure has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95%, 96%, 97%, or 99% or more amino acid sequence identity with natural P4HA.The P4HA used in the method for producing procollagen or collagen of the present disclosure may contain one or more amino acid substitutions, insertions, or deletions compared to natural P4HA.

[0072] In one aspect, the P4HA used in the method for producing procollagen or collagen of the present disclosure can be expressed as a fusion protein fused with a heterologous protein.Methods for producing heterologous proteins are known to those skilled in the art, and for example, a fusion protein of P4HA and a heterologous protein can be expressed by using a nucleic acid construct comprising a nucleic acid sequence encoding a fusion protein of P4HA and a heterologous protein.The manner in which P4HA and a heterologous protein are fused is not particularly limited, and the positional relationship between the nucleic acid sequence encoding part or all of P4HA and the nucleic acid sequence encoding part or all of the heterologous protein, the presence or absence of a linker sequence, etc. can be appropriately selected by those skilled in the art according to the purpose.

[0073] A nucleic acid construct containing a nucleic acid sequence encoding P4HA can be prepared by conventional chemical synthesis methods known to those skilled in the art to which this disclosure pertains, or by genetic engineering techniques. For example, it can be synthesized by synthesizing a cDNA having a naturally occurring sequence, and then using this cDNA as a template to introduce appropriate mutations using appropriate primers. Alternatively, the nucleic acid construct to be synthesized can be divided into several short regions, and multiple DNA fragments can be chemically synthesized, and the fragments can be linked using the known fusion PCR method to synthesize a DNA construct. An RNA construct can be prepared by transcription from the DNA construct.

[0074] Any method known to those skilled in the art to which the present disclosure pertains can be used to introduce a mutation into the sequence of a nucleic acid construct containing a nucleic acid sequence encoding P4HA. For example, those skilled in the art can use site-directed mutagenesis methods such as the Kunkel method and the gapped duplex method, overlap extension PCR, and QuikChange, but are not limited to these.

[0075] A nucleic acid construct comprising a nucleic acid sequence encoding P4HA may be DNA, RNA, or any analog thereof known to those skilled in the art to which the present disclosure pertains. For example, the nucleic acid construct of the present disclosure may contain unnatural bases, and may also contain a hydrocarbon chain or peptide chain as part thereof.

[0076] The step of expressing PH4A in a plant or isolated plant cells can be carried out using methods known in the technical field of the present disclosure. In one aspect, PH4A is transiently expressed in the method of producing procollagen or collagen of the present disclosure. Methods known in the technical field of the present disclosure can be used to transiently express PH4A in a plant or isolated plant cells. For example, transient expression of procollagen from a plant or isolated plant cells can be achieved by infecting a plant or isolated plant cell with a nucleic acid encoding PH4A as an expression vector. In one aspect, PH4A can be transiently expressed by transforming Agrobacterium tumefaciens with a vector plasmid and then infecting a plant or isolated plant cell with the Agrobacterium.

[0077] The type of collagen used in the method for producing procollagen or collagen of the present disclosure is not particularly limited, and by combining a nucleic acid construct encoding an appropriate α chain, any collagen can be produced, for example, type I collagen, type II collagen, type III collagen, etc. For example, collagen having a non-natural amino acid sequence or collagen fused with a heterologous peptide, as described below (Procollagen or collagen encoded by nucleic acid construct), can be produced.

[0078] The procollagen or collagen used in the method for producing collagen of the present disclosure has a naturally occurring or non-naturally occurring amino acid sequence. In one aspect, the procollagen or collagen used in the method for producing collagen of the present disclosure has an amino acid sequence identity of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95%, about 96% or more, about 97% or more, about 98% or more, or about 99% or more to a naturally occurring procollagen or collagen. The procollagen or collagen of the present disclosure may contain one or more amino acid substitutions, insertions, or deletions relative to a naturally occurring procollagen or collagen.

[0079] The procollagen or collagen used in the method for producing collagen of the present disclosure can be fused with a heterologous protein in place of the N-terminal proregion, thereby adding various functions. For example, fluorescent collagen can be produced by fusing GFP in place of the N-terminal proregion. The procollagen or collagen can be expressed and purified by any method known to those skilled in the art in the technical field to which the present disclosure pertains, and can be used for purposes such as analyzing chemical properties and physiological activity.

[0080] A nucleic acid construct containing a nucleic acid sequence encoding procollagen can be prepared by conventional chemical synthesis methods known to those skilled in the art to which this disclosure pertains, or by genetic engineering techniques. For example, it can be synthesized by synthesizing a cDNA having a naturally occurring sequence, and then using this cDNA as a template to introduce appropriate mutations using appropriate primers. Alternatively, the nucleic acid construct to be synthesized can be divided into several short regions, and multiple DNA fragments can be chemically synthesized, and the fragments can be ligated using the known fusion PCR method to synthesize a DNA construct. An RNA construct can be prepared by transcription from the DNA construct.

[0081] Any method known to those skilled in the art to which the present disclosure pertains can be used to introduce a mutation into the sequence of a nucleic acid construct containing a nucleic acid sequence encoding procollagen. For example, those skilled in the art can use site-directed mutagenesis methods such as the Kunkel method and the gapped duplex method, overlap extension PCR, and QuikChange, but are not limited to these.

[0082] A nucleic acid construct comprising a nucleic acid sequence encoding procollagen may be DNA, RNA, or any analog thereof known to those skilled in the art to which the present disclosure pertains. For example, the nucleic acid construct of the present disclosure may contain unnatural bases, and may also contain a hydrocarbon chain or peptide chain as part thereof.

[0083] In one aspect, in the method of producing procollagen or collagen of the present disclosure, the procollagen may be encoded by a nucleic acid construct encoding PH4B, or may be encoded by a separate nucleic acid construct.

[0084] The step of expressing procollagen in a plant or isolated plant cells can be carried out using methods known in the technical field of the present disclosure. In one embodiment, procollagen is transiently expressed. Methods known in the technical field of the present disclosure can be used to transiently express procollagen in a plant or isolated plant cells. For example, transient expression of procollagen from a plant or isolated plant cell can be achieved by infecting the plant or isolated plant cell with a nucleic acid encoding procollagen as an expression vector. In one embodiment, collagen can be transiently expressed by transforming Agrobacterium tumefaciens with a vector plasmid and then infecting the Agrobacterium into a plant or isolated plant cell.

[0085] In one aspect, the method for producing procollagen or collagen of the present disclosure includes a step of expressing an enzyme useful for stable expression of collagen in a plant body or isolated plant cell in a plant body or isolated plant cell. For example, LH3 or the like can be used as an enzyme that enhances collagen stability. Methods known in the technical field of the present disclosure can be used to express these enzymes in a plant body or isolated plant cell. For example, expression of these enzymes can be achieved by transforming nucleic acid sequences encoding them into a plant body or isolated plant cell. Enzymes derived from various organisms can be used as these enzymes, for example, enzymes derived from nematodes, mice, humans, etc. The nucleic acid sequences encoding these enzymes may be contained in a nucleic acid construct encoding PH4B or a nucleic acid construct encoding procollagen, or may be contained in different nucleic acid constructs.

[0086] Plants that can be used in the procollagen or collagen production method of the present disclosure include, for example, Nicotiana plants, bryophytes (e.g., P. patens), potatoes (e.g., S. tuberosum), grasses (e.g., O. sativa), Brassicaceae plants, and lettuce, with Nicotiana plants being preferred. Examples of Nicotiana plants include, but are not limited to, N. benthamiana, N. tabacum, and N. excelsior.

[0087] (Procollagen or Collagen Produced by the Production Method of the Present Disclosure) In one aspect, the present disclosure provides procollagen or collagen produced using a plant or isolated plant cell comprising a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity.

[0088] In one aspect, the present disclosure provides procollagen or collagen produced by a method for producing procollagen or collagen, the method comprising the steps of: A) providing a plant or isolated plant cell containing a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell.

[0089] The method for producing procollagen or collagen of the present disclosure is carried out as described above (the method for producing procollagen or collagen using a plant or isolated plant cells containing a nucleic acid construct of the present disclosure). The procollagen or collagen produced by the production method of the present disclosure has excellent thermal stability and is suitable for use in, for example, the fields of medicine, cosmetics, or food.

[0090] (Composition Comprising Procollagen or Collagen Produced by the Production Method of the Present Disclosure) In one aspect, the present disclosure provides a composition comprising procollagen or collagen produced using a plant or isolated plant cell comprising a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity.

[0091] In one aspect, the present disclosure provides a composition comprising procollagen or collagen produced by a method for producing procollagen or collagen, the method comprising the steps of: A) providing a plant or isolated plant cell comprising a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) and transiently expressing procollagen in the plant or isolated plant cell.

[0092] In one aspect, the composition of the present disclosure is not limited in form and may be provided in the form of, for example, a pharmaceutical composition, a cosmetic composition, or a food composition, and may contain, in addition to procollagen or collagen, carriers, excipients, etc. that are acceptable for compositions in these fields. In the composition of the present disclosure, the procollagen or collagen may be in a multimeric state having a fibrous or network structure containing multiple procollagen or collagen molecules, further resulting from an intermolecular crosslinking treatment. The amount of collagen contained in the composition of the present disclosure is not particularly limited and may be adjusted as appropriate depending on the dosage form, purpose, etc.

[0093] Pharmaceutical compositions containing the procollagen or collagen of the present disclosure include those in which the composition is a pharmaceutical product itself, and those that are raw materials or intermediate products used in the production of pharmaceutical products. The form of the pharmaceutical composition of the present disclosure is not particularly limited, and may be solid, powder, granules, or liquid. When the pharmaceutical composition of the present disclosure is administered to a subject, its dosage form is not particularly limited, and it may be provided as, for example, tablets, fine granules, pills, lozenges, capsules, or raw materials or intermediate products thereof.

[0094] The pharmaceutical compositions of the present disclosure include compositions that are pharmaceuticals for regenerative medicine, or raw materials or intermediate products for the production thereof. For example, the pharmaceutical compositions of the present disclosure may be provided as a sheet-shaped composition and used to promote tissue regeneration within or on the surface of a subject's body, or as a scaffold for culturing sheet-shaped tissue. In one aspect, the pharmaceutical compositions of the present disclosure may be provided as a material composition for constructing a regenerative medicine product having a three-dimensional structure using 3D printing technology, for example, as a raw material for bioink.

[0095] In one aspect, pharmaceutical compositions of the present disclosure include compositions that are implantable medical devices or raw materials or intermediate products in the manufacture thereof. These compositions can be used for the prevention or treatment of diseases or disorders, cosmetic purposes, etc. Non-limiting examples of such compositions include wound dressings, artificial skin, artificial blood vessels, catheters and other devices for removing or delivering fluids to patients, artificial hearts, artificial kidneys, and orthopedic pins, plates, and implants. For example, pharmaceutical compositions of the present disclosure can be used as 3D bioprinted post-mastectomy implants or fillers for cosmetic surgery.

[0096] Cosmetic compositions containing the procollagen or collagen of the present disclosure include those that are the cosmetic itself and those that are raw materials or intermediate products used in the production of cosmetics. The form of the cosmetic composition of the present disclosure is not particularly limited, and may be solid, powder, granules, or liquid. The cosmetic composition of the present disclosure may be applied topically or administered orally, and may be provided, for example, as topical preparations such as ointments, lotions, creams, microemulsions, gels, oils, and solutions; or oral preparations such as tablets, fine granules, pills, lozenges, and capsules; or as raw materials or intermediate products thereof.

[0097] The food compositions containing procollagen or collagen of the present disclosure include those in which the composition is the food itself, and those that are raw materials or intermediate products in the production of food. Such foods include health foods, functional foods, foods for specified health uses, and foods for the sick, and also include feed when used for animals other than humans. The form of the food is not particularly limited, and may be solid or liquid. The animals other than humans are not particularly limited, and various animals such as mammals, birds, reptiles, amphibians, fish, and insects can be used as targets. Specific types of food include, for example, beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and dairy drinks, as well as concentrated liquids and powders for adjusting these beverages; frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; baked goods such as candy, chewing gum, candies, gummies, chewing gum, caramel, chocolate, candy tablets, snacks, and biscuits, as well as confectioneries such as jelly, jam, and cream; processed seafood and livestock foods such as kamaboko, hamburger steak, ham, and sausage; dairy products such as processed milk, fermented milk, yogurt, butter, and cheese; oils and fats and oil-based foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; condiments such as sauces and dressings; soups, stews, curries, bread, jams, salads, side dishes, and pickles, but are not limited to these.

[0098] (Method for preventing death of a plant or isolated plant cell) In one aspect, the present disclosure provides a method for preventing death of a plant or isolated plant cell that contains a nucleic acid encoding collagen or procollagen.

[0099] In one aspect, the method of the present disclosure for preventing the withering of a plant or isolated plant cell comprises: A) introducing into the plant or isolated plant cell a nucleic acid encoding collagen or procollagen; and B) introducing into the plant or isolated plant cell a nucleic acid construct having an expression control element functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B is deficient in protein disulfide isomerase (PDI) activity, and a nucleic acid encoding P4HA.

[0100] In the method of the present disclosure, the order of step A) and step B) is not particularly limited, and step A) may be followed by step B), or step B) may be followed by step A). ​​In one embodiment, step A) and step B) may be performed simultaneously.

[0101] In one aspect, the plant or isolated plant cell comprising a nucleic acid encoding collagen or procollagen in step A) above is suitably prepared by the method described above (Plant or isolated plant cell comprising a nucleic acid construct of the present disclosure). For example, the plant or isolated plant cell may be obtained by introducing a nucleic acid construct, or by growing or propagating the treated plant or plant cell. In one aspect, the plant or isolated plant cell comprising a nucleic acid construct of the present disclosure may be a subculture of a plant that has been introduced with a nucleic acid construct.

[0102] In one embodiment, the plant or isolated plant cell in the method of the present disclosure transiently expresses collagen or procollagen. The transient expression of collagen or procollagen can be achieved by using the method described above (Method for producing procollagen or collagen using a plant or isolated plant cell containing a nucleic acid construct of the present disclosure). For example, collagen or procollagen can be transiently expressed in the plant or isolated plant cell using a transformation vector or the like.

[0103] In one aspect, the nucleic acid construct described above (Nucleic acid construct of the present disclosure) can be suitably used as the nucleic acid construct in step B) above, which has an expression control element functional in plant cells and a sequence encoding prolyl 4-hydroxylase B subunit (PH4B), wherein the encoded PH4B lacks protein disulfide isomerase (PDI) activity.

[0104] The plants to which the method for preventing the withering of plants or isolated plant cells of the present disclosure can be applied are not particularly limited, but are preferably applied to plants used in the above-mentioned method for producing procollagen or collagen using plants or isolated plant cells containing the nucleic acid construct of the present disclosure. Examples of such plants include Nicotiana plants, bryophytes (e.g., P. patens), potato (e.g., S. tuberosum), grasses (e.g., O. sativa), Brassicaceae plants, and lettuce, with Nicotiana plants being preferred. Examples of Nicotiana plants include, but are not limited to, Nicotiana benthamiana, Nicotiana tabacum, and Nicotiana excelsior.

[0105] In one embodiment, the plant or isolated plant cell in the method of the present disclosure transiently expresses P4HA and P4HB lacking PDI activity. For this transient expression, the method described above (Method for producing procollagen or collagen using a plant or isolated plant cell containing the nucleic acid construct of the present disclosure) for transient expression of P4HA and P4HB can be suitably used.

[0106] In one aspect, the method for preventing the withering of a plant or isolated plant cells further comprises, after step A) and step B), a step of cultivating or culturing the plant or isolated plant cells for about 6 to 15 days, 7 to 15 days, 8 to 15 days, 9 to 15 days, or 10 to 15 days.

[0107] Prevention of withering of a plant or isolated plant cells can be confirmed by a method common in the art. In one aspect, when comparing the appearance of a plant or isolated plant cells expressing procollagen or collagen (control) to which the method of the present disclosure has not been applied with the appearance of a plant or isolated plant cells to which the method of the present disclosure has been applied, it can be confirmed that the withering of a plant or isolated plant cells has been prevented by confirming that signs of withering, such as necrosis in at least a portion of the plant or plant cells, are reduced or eliminated compared to the control.

[0108] The present disclosure will be described in more detail below using examples, but these examples are merely illustrative and do not limit the scope of the present disclosure to the scope of the examples.

[0109] (Example 1) Construction of modified P4HB

[0110] 1. Vector Construction 1-1. Construction of Collagen Gene Expression Cassette A synthetic gene encoding the collagen alpha-1 (I) chain (Col1, DNA sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4) fused to the barley amylase signal peptide (Barley AMY 1.2 Signal Peptide, DNA sequence: SEQ ID NO: 1, amino acid sequence: SEQ ID NO: 2) was cloned into an expression cassette consisting of the CaMV 35S promoter (SEQ ID NO: 5), Arabidopsis thaliana ADH-derived 5'-UTR (SEQ ID NO: 6), and HSP terminator (SEQ ID NO: 8) (Figure 1).

[0111] We predicted the changes in thermal stability due to mutations to cysteine ​​residues and confirmed that, of the two cysteine ​​residues in each active center, mutations to the N-terminal side resulted in a smaller overall degree of destabilization after mutation. Furthermore, in addition to serine residues, alanine and histidine residues, which have relatively low destabilizing effects, were also considered as potential mutation targets. Furthermore, considering the possibility that uncomplexed P4HB alone may adversely affect plants, we also investigated the expression of fusion proteins of P4HA and modified P4HB. Furthermore, we investigated the expression of fusion proteins of P4HA with water-soluble partners, MBP and GFP. In addition to human P4HAB, chimeras of nematode P4HA and mouse P4HB were used for the study.

[0112] 1-2. Construction of modified enzyme expression vectors Synthetic genes encoding human P4HA (DNA sequence: SEQ ID NO:9, amino acid sequence: SEQ ID NO:10), human P4HB (DNA sequence: SEQ ID NO:11, amino acid sequence: SEQ ID NO:12), nematode P4HA (DNA sequence: SEQ ID NO:13, amino acid sequence: SEQ ID NO:14), and mouse P4HB (DNA sequence: SEQ ID NO:15, amino acid sequence: SEQ ID NO:16) fused to the barley amylase signal peptide (DNA sequence: SEQ ID NO:1, amino acid sequence: SEQ ID NO:2) were cloned into expression cassettes consisting of the CaMV 35S promoter (SEQ ID NO:5) and the Arabidopsis thaliana ADH-derived 5'-UTR (SEQ ID NO:6) or TMV omega sequence (SEQ ID NO:7), and the HSP-derived terminator (SEQ ID NO:8) (Figure 2). Furthermore, to eliminate the function of one or both of the two PDI active centers (human P4HB: positions 53-56 and 397-400), we constructed modified P4HBs in which the two cysteine ​​residues present in each active center were altered to alanine, serine, or histidine residues (Figure 5).

[0113] When selecting the substituted amino acid residues, the online tool Site-Directed Mutator (SDM2) http: / / marid.bioc.cam.ac.uk / sdm2 / prediction (Nucleic Acids Res. 2011, Vol. 39, W215-W222, doi: 10.1093 / nar / gkr363, Scientific Reports, 2021, Vol. 11, Article number: 10202) was used to predict the effect of amino acid substitution on the thermal stability of the P4HB protein, and the N-terminal cysteine ​​residue was substituted, which was predicted to have a small degree of destabilization after mutagenesis. The prediction results using human PH4B are shown in the table below.

[0114] Expression cassettes for fusion proteins of P4HA and modified P4HB, or P4HA and a water-soluble protein (MBP or GFP) were constructed (Figure 3). The stop codon of P4HA was removed, and the gene for modified P4HB or other fusion proteins was linked via an appropriate linker sequence (GSGSGRITMLSRALLCLALAWAARVGA).

[0115] A synthetic gene (DNA sequence: SEQ ID NO: 17, amino acid sequence: SEQ ID NO: 18) encoding the RNA silencing suppressor p19 (UniProtKB-P50628) derived from Tomato bushy stunt virus was cloned into an expression cassette consisting of the CaMV 35S promoter (SEQ ID NO: 5), the Arabidopsis thaliana ADH-derived 5'-UTR (SEQ ID NO: 6), and the HSP-derived terminator (SEQ ID NO: 8) (Figure 4).

[0116] The constructed expression cassettes for each gene were cloned into the multicloning site of the pRI 201-AN plant transformation vector (Takara Bio) in the following combinations: (1) Col1 + p19 (2) Col1 + P4HA + modified P4HB + p19 (3) Col1 + P4HA modified P4HB fusion + p19 (4) Col1 + P4HA modified MBP fusion + p19 (5) Col1 + P4HA modified GFP fusion + p19

[0117] (Example 2) Effect of Improving Collagen Stability 2-1 Transformation of Agrobacterium Using the plasmids derived from the pRI201-AN plant transformation vector prepared above, Agrobacterium strain GV3101 was transformed by the freeze-thaw method. Agrobacterium transformed with each plasmid was cultured overnight at 28°C with shaking in Select Alternative Protein Source (APS) medium (Becton, Dickinson and Company) containing 50 μg / ml kanamycin, and grown until the OD600 reached 4.5 to 5.5. The Agrobacterium culture was centrifuged, and the pellet was resuspended in infiltration buffer (10 mM MgCl ２ The bacterial suspension was resuspended in 10 mM MES (pH 5.6) and the concentration of the bacterial suspension was adjusted to an OD600 of 0.4, and infiltration was carried out.

[0118] 2-2 Transient Expression in Plants Nicotiana benthamiana plants were grown from seeds in plug trays filled with commercially available coconut shell substrate. The plants were grown in a greenhouse under a 16-hour / 8-hour photoperiod and a temperature schedule of 25°C daytime / 20°C nighttime. Four weeks after sowing, the plants were agroinfiltrated and inoculated with vectors that co-express collagen α1 and P4HA, as well as P4HB with one or both PDI active centers modified. Immediately before infiltration, the apical bud was removed by pinching from the plants.

[0119] After infiltration, the plants were incubated for six days and only the leaves infiltrated with Agrobacterium were harvested. Observation of the degree of necrosis at harvest revealed that, regardless of origin or whether fusion occurred, plants inoculated with vectors in which one of the two PDI active centers of P4HB remained tended to show severe necrosis, indicating that modification of both PDI active centers is desirable. Figure 6 shows the condition of the plants after six days of incubation. A shows a plant inoculated with P4HB in which only one PDI active center had been modified, and B shows a plant inoculated with P4HB in which both PDI active centers had been modified.

[0120] 2-3 Confirmation of collagen heat resistance Total soluble protein was extracted by adding 3 volumes of 50 mM Tris (pH 7.4), 0.15 M NaCl to approximately 0.1 g of frozen and crushed leaves, grinding them, and centrifuging them at 20,000 g for 5 minutes at 4°C. The presence or absence of collagen expression was confirmed by SDS-PAGE (Figure 7). In Figure 7, the arrow indicates the collagen α1 band. Collagen bands were confirmed for all vectors except for Lane 15 and Lane 16 (C. elegans P4HA / mouse P4HB_C-S fusion).

[0121] The thermal stability of the collagen triple helix structure was confirmed by heat denaturation and trypsin digestion of the extract. 25 μL of the extract was incubated at 33°C for 20 minutes (heat treatment) and then immediately placed on ice. Trypsinization was performed by adding 1 μL of trypsin-EDTA to each sample and incubating at 22°C for 20 minutes. The reaction was stopped by adding 2x sample buffer containing DTT and tested by standard Western blotting procedures using anti-collagen α1 antibody. To further analyze the heat resistance, some samples were further heat-treated under gradient conditions, and the degree of degradation at each temperature was compared.

[0122] The results of trypsin treatment after heat treatment at 33°C are shown in Figure 8. Several collagen degradation product bands with different degrees of degradation were observed for some vectors. Under the same conditions, collagen was completely degraded in the extract from plants expressing collagen α1 alone (lane 2), suggesting that the heat resistance of collagen was improved in plants expressing modified P4HB. However, in plants expressing P4HB with a modification of the histidine residue in the PDI active center (lanes 5, 6, 17, and 18), a clear collagen band was detected in SDS-PAGE (Figure 7), but was not detected after trypsin treatment, suggesting that P4HB function has likely been lost.

[0123] An additional test was performed on some of the extracts that showed favorable results, with heat treatment under gradient conditions followed by trypsin treatment. The results are shown in Figures 9 and 10. In plants expressing the human P4HA / human P4HB_NC-AA fusion protein, plants co-expressing human P4HA and human P4HB_NC-SS, and plants expressing the nematode P4HA / mouse P4HB_NC-SS fusion protein, undegraded collagen was detected at temperatures below 24.26°C. In contrast, collagen α1 expression alone resulted in collagen degradation by trypsin treatment even at low heat treatment temperatures. This experiment demonstrates that plants expressing modified P4HB have improved collagen resistance to trypsin after heat treatment.

[0124] 2-4 Investigation of fusion proteins of P4HA and other water-soluble proteins A fusion protein of P4HA and water-soluble proteins (MBP, GFP) was co-expressed with collagen α1, and the results of SDS-PAGE and Western blotting after heat treatment at 33°C and trypsin treatment are shown in Figure 11. Collagen was detected in SDS-PAGE but not in Western blotting after trypsin treatment, indicating that the fusion protein of P4HA and water-soluble proteins does not function sufficiently as a modifying enzyme for collagen expressed in plants.

[0125] The above examples have confirmed that by co-expressing P4HA and P4HB lacking PDI activity, or using a fusion protein of these, as a modifying enzyme to be co-expressed when transiently expressing collagen protein in a plant expression system, it is possible to suppress necrosis of the plant body while enabling post-translational modification of plant-expressed collagen, and to obtain collagen with excellent thermal stability.

[0126] (Sequence List)

Claims

1. A nucleic acid construct having expression control elements functional in a plant cell and a sequence encoding prolyl 4-hydroxylase B subunit (P4HB), wherein the encoded P4HB is deficient in protein disulfide isomerase (PDI) activity.

2. The nucleic acid construct according to claim 1, wherein the P4HB encoded by the nucleic acid sequence has the function of post-translationally modifying procollagen or collagen in a plant cell expressing P4HA.

3. The nucleic acid construct according to claim 1, wherein the P4HB encoded by the nucleic acid sequence has an amino acid substitution in the PDI active center of the subunit.

4. The nucleic acid construct of claim 1, wherein the P4HB encoded by said nucleic acid sequence is fused to a prolyl 4-hydroxylase A subunit.

5. The nucleic acid construct of claim 1, wherein the plant is a Nicotiana plant.

6. A composition comprising the nucleic acid construct of claim 1.

7. A plant or isolated plant cell comprising the nucleic acid construct of claim 1.

8. Use of a nucleic acid construct according to any one of claims 1 to 5, or a composition according to claim 6, or a plant or isolated plant cell according to claim 7 in a method for producing procollagen or collagen from a plant or an isolated plant cell.

9. A method for producing procollagen or collagen, comprising the steps of: A) preparing a plant or an isolated plant cell comprising the nucleic acid construct according to any one of claims 1 to 5, a nucleic acid encoding P4HA, and a nucleic acid encoding collagen or procollagen; and B) expressing P4HB and prolyl 4-hydroxylase A subunit (P4HA) in the plant or isolated plant cell, and transiently expressing procollagen.

10. The method according to claim 9, further comprising the step of: C) separating the procollagen or collagen secreted outside the cells by the step B.

11. Procollagen or collagen produced by the method of claim 9.

12. A method for preventing the withering of a plant or an isolated plant cell, comprising: A) introducing into the plant or isolated plant cell a nucleic acid encoding collagen or procollagen; and B) introducing into the plant or isolated plant cell the nucleic acid construct according to any one of claims 1 to 5 and a nucleic acid encoding P4HA.

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